Microglia, the resident immune cells of the central nervous system, normally exist in a "resting" state with small cell bodies and ramified processes. In cases of injury, such as rupture of small blood vessels, microglia extend their processes to the site of injury and appear to clear cellular debris by phagocytosis. This directional process extension is mediated by ATP released from damaged cells and activation of P2Y12 receptors on microglia. However, in prolonged injury and in many neurodegenerative conditions microglia assume an amoeboid "activated" phenotype characterized by larger cell bodies, stunted processes, and release of pro-inflammatory cytokines. Surprisingly, ATP causes process retraction and migration away from its source in the lipopolysaccharide (LPS) model of activated microglia in vitro. This switch in response is thought to be the consequence of P2Y12 receptor downregulation and concurrent A2A receptor upregulation that occur with microglial activation. Here, we hypothesize that A2A receptor activation contributes to the pro-inflammatory phenotype of activated microglia by increasing cytokine and reactive oxygen species (ROS) secretion in vitro and altering the ability of microglia to respond to injury in vivo. In order to test this hypothesis, we propose to examine (1) if A2A receptor activation changes the pattern of cytokine and/or ROS secretion by activated microglia;and (2) whether resting and activated microglia respond differently to neuronal injury. The effect of A2A receptor activation on the production of representative cytokines and ROS will be examined in order to determine the role that A2A receptors might play in modifying important microglial functions. Then, we will examine the effects of an adenosine gradient on microglial motility in vitro. Finally, we will perform two photon imaging of GFP-expressing microglia in vivo to determine if activation of A2A receptors on microglia modulates their response to neuronal damage. The successful completion of the proposed experiments will elucidate the function of A2A receptors in activated microglia and provide a better understanding of how targeting these receptors for therapeutic purposes might affect microglia in the CNS. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to study a mechanism of regulation of the brain's own immune system. The results from the research might have implications on the role of the immune system in the development of neurodegenerative diseases, and provide a way modulate disease progression. In addition, the research will provide new insights on how common environmental agents such as caffeine might affect the immune cells of the brain, and thus brain pathologies.